In-depth characterization of intratumoral heterogeneity in refractory B-cell non-Hodgkin lymphoma through the lens of a Research Autopsy Program.

Intratumoral heterogeneity (ITH) provides the substrate for tumor evolution and treatment resistance, yet is remarkably understudied in lymphoma, due to the often limited amount of tissue that gets sampled during the routine diagnostic process, generally from a single nodal or extranodal site. Furthermore, the trajectory of how lymphoma, and especially non-Hodgkin lymphoma, spreads throughout the human body remains poorly understood. Here, we present a detailed characterization of ITH by applying whole-genome sequencing to spatially separated tumor samples harvested at the time of autopsy (n=24) and/or diagnosis (n=3) in three patients presenting with refractory B-cell non-Hodgkin lymphoma. Through deconvolution of bulk samples into clonal mixtures and inference of phylogenetic trees, we found evidence that polyclonal seeding underlies tumor dissemination in lymphoma. We identify mutation signatures associated with ancestral and descendant clones. In our series of patients with highly refractory lymphoma, the determinants of resistance were often harbored by founding clones, although there was also evidence of positive selection of driver mutations, likely under the influence of therapy. Lastly, we show that circulating tumor DNA is suitable for the detection of ancestral mutations but may miss a significant proportion of private mutations that can be detected in tissue. Our study clearly shows the existence of intricate patterns of regional and anatomical evolution that can only be disentangled through multi-regional tumor tissue profiling.

Clinical significance of clonal hematopoiesis in the setting of autologous stem cell transplantation for lymphoma.

Autologous stem cell transplantation (ASCT) remains a key therapeutic strategy for treating patients with relapsed or refractory non-Hodgkin and Hodgkin lymphoma. Clonal hematopoiesis (CH) has been proposed as a major contributor not only to the development of therapy-related myeloid neoplasms but also to inferior overall survival (OS) in patients who had undergone ASCT. Herein, we aimed to investigate the prognostic implications of CH after ASCT in a cohort of 420 lymphoma patients using ultra-deep, highly sensitive error-correction sequencing. CH was identified in the stem cell product samples of 181 patients (43.1%) and was most common in those with T-cell lymphoma (72.2%). The presence of CH was associated with a longer time to neutrophil and platelet recovery. Moreover, patients with evidence of CH had inferior 5-year OS from the time of first relapse (39.4% vs. 45.8%, p = .043) and from the time of ASCT (51.8% vs. 59.3%, p = .018). The adverse prognostic impact of CH was not due to therapy-related myeloid neoplasms, the incidence of which was low in our cohort (10-year cumulative incidence of 3.3% vs. 3.0% in those with and without CH, p = .445). In terms of specific-gene mutations, adverse OS was mostly associated with PPM1D mutations (hazard ratio (HR) 1.74, 95% confidence interval (CI) 1.13-2.67, p = .011). In summary, we found that CH is associated with an increased risk of non-lymphoma-related death after ASCT, which suggests that lymphoma survivors with CH may need intensified surveillance strategies to prevent and treat late complications.

GNAS knockout potentiates HDAC3 inhibition through viral mimicry-related interferon responses in lymphoma.

Despite selective HDAC3 inhibition showing promise in a subset of lymphomas with CREBBP mutations, wild-type tumors generally exhibit resistance. Here, using unbiased genome-wide CRISPR screening, we identify GNAS knockout (KO) as a sensitizer of resistant lymphoma cells to HDAC3 inhibition. Mechanistically, GNAS KO-induced sensitization is independent of the canonical G-protein activities but unexpectedly mediated by viral mimicry-related interferon (IFN) responses, characterized by TBK1 and IRF3 activation, double-stranded RNA formation, and transposable element (TE) expression. GNAS KO additionally synergizes with HDAC3 inhibition to enhance CD8+ T cell-induced cytotoxicity. Moreover, we observe in human lymphoma patients that low GNAS expression is associated with high baseline TE expression and upregulated IFN signaling and shares common disrupted biological activities with GNAS KO in histone modification, mRNA processing, and transcriptional regulation. Collectively, our findings establish an unprecedented link between HDAC3 inhibition and viral mimicry in lymphoma. We suggest low GNAS expression as a potential biomarker that reflects viral mimicry priming for enhanced response to HDAC3 inhibition in the clinical treatment of lymphoma, especially the CREBBP wild-type cases.

Identification of genetic subtypes in follicular lymphoma.

Follicular lymphoma (FL) exhibits considerable variability in biological features and clinical trajectories across patients. To dissect the diversity of FL, we utilized a Bernoulli mixture model to identify genetic subtypes in 713 pre-treatment tumor tissue samples. Our analysis revealed the existence of five subtypes with unique genetic profiles that correlated with clinicopathological characteristics. The clusters were enriched in specific mutations as follows: CS (CREBBP and STAT6), TT (TNFAIP3 and TP53), GM (GNA13 and MEF2B), Q (quiescent, for low mutation burden), and AR (mutations of mTOR pathway-related genes). The subtype Q was enriched for patients with stage I disease and associated with a lower proliferative history than the other subtypes. The AR subtype was unique in its enrichment for IgM-expressing FL cases and was associated with advanced-stage and more than 4 nodal sites. The existence of subtypes was validated in an independent cohort of 418 samples from the GALLIUM trial. Notably, patients assigned to the TT subtype consistently experienced inferior progression-free survival when treated with immunochemotherapy. Our findings offer insight into core pathways distinctly linked with each FL cluster and are expected to be informative in the era of targeted therapies.

Sarcocystis bovifelis in Raw Hamburgers Marketed in Hamadan City, Western Iran.

Background: We aimed to evaluate Sarcocystis contamination in conventional and industrial raw beef burger samples from butcheries and retail stores in Hamadan, western Iran. Methods: Overall, 80 samples including 30 conventional and 50 industrial hamburgers were randomly obtained from different butcheries and supermarkets. All specimens were studied by digestion method following microscopic examination. Samples' genomic ribosomal DNA were amplified and nucleotide sequences were analyzed by BLAST for comparison with the sequences in the gene bank of the NCBI. Results: Sarcocystis bradyzoites were detected in 46 of 80 (57.6%) samples. Positive specimens were included as 46 (57.6%) and 30 (37.5%) by digestion and molecular method, respectively. Differences between two studied (digestion and molecular) methods was statistically significant (P=0.00). Twenty-six (86.5 %) of 30 conventional beef burgers and 20 (40%) of 50 industrial burgers were positive for Sarcocystis sp. by digestion method. There was a significant difference between Sarcocystis infested conventional and industrial beef burgers (P=0.01). Conclusion: The parasitic contamination of beef burgers implied a high level of infection in cattle. Felids as the definitive hosts for S. bovifelis urged on the improvement of the hygienic conditions of keeping and feeding livestock in order to reduce the infection. Molecular techniques confirm species in meat products with high sensitivity and distinguish it from human species.

DNA Methylation-Based Classification of Small B-Cell Lymphomas: A Proof-of-Principle Study.

Although most small B-cell lymphomas (SBCLs) can be diagnosed using routine methods, challenges exist. For example, marginal zone lymphomas (MZLs) can be difficult to rule-in, in large part because no widely-used, sensitive, and specific biomarker is available for the marginal zone cell of origin. In this study, it was hypothesized that DNA methylation array profiling can assist with the classification of SBCLs, including MZLs. Extramedullary SBCLs, including challenging cases, were reviewed internally for pathology consensus and profiled. By combining the resulting array data set with data sets from other groups, a set of 26 informative probes was selected and used to train machine learning models to classify 4 common SBCLs: chronic lymphocytic leukemia/small lymphocytic lymphoma, follicular lymphoma, mantle cell lymphoma, and MZL. Prediction probability cutoff was used to separate classifiable from unclassifiable cases, and show that the trained model was able to classify 95% of independent test cases (n = 264/279). The concordance between model predictions and pathology diagnoses was 99.6% (n = 262/263) among classifiable test cases. One validation reference test case was reclassified based on model prediction. The model was also used to predict the diagnoses of two challenging SBCLs. Although the differential examined and data on difficult cases are limited, these results support accurate methylation-based classification of SBCLs. Furthermore, high specificities of predictions suggest that methylation signatures can be used to rule-in MZLs.

Combined EZH2 Inhibition and IKAROS Degradation Leads to Enhanced Antitumor Activity in Diffuse Large B-cell Lymphoma.

PURPOSE: The efficacy of EZH2 inhibition has been modest in the initial clinical exploration of diffuse large B-cell lymphoma (DLBCL), yet EZH2 inhibitors are well tolerated. Herein, we aimed to uncover genetic and pharmacologic opportunities to enhance the clinical efficacy of EZH2 inhibitors in DLBCL. EXPERIMENTAL DESIGN: We conducted a genome-wide sensitizing CRISPR/Cas9 screen with tazemetostat, a catalytic inhibitor of EZH2. The sensitizing effect of IKZF1 loss of function was then validated and leveraged for combination treatment with lenalidomide. RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing analyses were performed to elucidate transcriptomic and epigenetic changes underlying synergy. RESULTS: We identified IKZF1 knockout as the top candidate for sensitizing DLBCL cells to tazemetostat. Treating cells with tazemetostat and lenalidomide, an immunomodulatory drug that selectively degrades IKAROS and AIOLOS, phenocopied the effects of the CRISPR/Cas9 screen. The combined drug treatment triggered either cell-cycle arrest or apoptosis in a broad range of DLBCL cell lines, regardless of EZH2 mutational status. Cell-line-based xenografts also showed slower tumor growth and prolonged survival in the combination treatment group. RNA-seq analysis revealed strong upregulation of interferon signaling and antiviral immune response signatures. Gene expression of key immune response factors such as IRF7 and DDX58 were induced in cells treated with lenalidomide and tazemetostat, with a concomitant increase of H3K27 acetylation at their promoters. Furthermore, transcriptome analysis demonstrated derepression of endogenous retroviruses after combination treatment. CONCLUSIONS: Our data underscore the synergistic interplay between IKAROS degradation and EZH2 inhibition on modulating epigenetic changes and ultimately enhancing antitumor effects in DLBCL.